The goal of this project is to determine how elevated levels of homocysteine (Hcy) (hyperhomocysteinemia, HHcy)) contribute to retinal neurovascular disease. HHcy is implicated in retinal vascular occlusive disease, optic neuropathies (e.g. pseudoexfoliation glaucoma), macular degeneration, and diabetic retinopathy. Mild HHcy occurs in ~5%-12% of the population; hence understanding how HHcy contributes to retinal disease is critically important. Hcy is a key intermediate in methionine metabolism. HHcy is due to genetic defects involving cystathionine synthase (CBS), methylenetetrahydrofolate reductase (MTHFR) or methionine synthase as well as vitamin deficiencies (folate/B12/B6). During the previous funding period we analyzed retinas of mice with endogenous HHcy due to deficiency/lack of CBS and observed profound loss of visual function, marked disruption of retinal neuronal layers and florid vasculopathy in cbs-/- mice, and a milder phenotype with vascular alterations, progressive ganglion cell loss, dysregulation of axonal mitochondrial dynamics and delayed visual evoked potentials in cbs+/- mice. These data suggest that HHcy compromises retinal structure/function, the severity of which correlates with Hcy levels; and that HHcy alters retinal vasculature initially, subsequently triggering retinal neuron death. The phenotype may be due to increased ER stress, oxidative stress, and/or excitotoxicity; however since CBS irreversibly converts Hcy to cystathionine via the transsulfuration pathway leading to formation of cysteine, which is then converted to glutathione (GSH), hydrogen sulfide (H2S), and/or taurine, the observed disrupted retinal structure/function may be due to decreased levels of these downstream factors critical for retinal health. To investigate this, we will analyze retinas of mice lacking/deficient in MTHFR. mthfr mice have a defect in the remethylation pathway with concomitant HHcy; however their transsulfuration pathway is intact. Using these models we can dissect the role of HHcy in retina. Upon completion of the studies we will be poised to investigate whether folate/B12/B6 supplementation reverses/retards retinal neurovasculopathy associated with HHcy due to mutations in CBS or mthfr. There are three aims proposed for this study: Aim 1: Test the hypotheses that (A) HHcy compromises retinal structure/function regardless whether due to a defect in the transsulfuration or remethylation pathway; and (B) severe HHcy alters retinal vasculature initially, which subsequently triggers widespread death of retinal neurons (vasculopathy precedes neuropathy) while mild HHcy induces modest vascular changes disrupting only the inner retina; Aim 2: Test the hypothesis that HHcy due to CBS deficiency induces retinal neurovascular disease not only by inducing oxidative stress, excitotoxicity, and/or ER stress, but also by altering levels of GSH, H2S and taurine, whereas HHcy due to MTHFR deficiency induces retinal neurovascular disease through the oxidative stress, excitotoxicity, and/or ER stress pathways; Aim 3: Test the hypothesis that folate/B12/B6 supplementation will retard/reverse HHcy-induced retinal neurovasculopathy.